This invention relates to a system for obtaining spectroscopically resolved images of materials, and particularly, for obtaining such images using spatial encoding with a two-dimensional image detector.
There are numerous instances in which spectroscopically resolved images from samples such as those observed under a microscope are desired. One such application is Raman spectroscopy. Raman spectroscopy is a technique which utilizes inelastic light scattering phenomena, which is commonly used to obtain vibrational spectra of a sample. A high intensity monochromatic beam impinges on the sample and a small fraction (about 1 in 10.sup.7) of the photons is scattered at (optical) frequencies which differ from the incident (optical) frequency by vibrational frequencies of the sample molecules. Most of this inelastic scattering occurs with loss of photon energy (red spectral shift) and it is this red-shifted scattered light which is usually collected as a Raman signal. Since the early 1960's lasers have been the preferred light source for Raman scattering. Many kinds of lasers have been employed. At present the most common ones are argon ion (457-514 nm), Nd-YAG (1.06 microns and 532 nm) and krypton ion (647 and 752 nm). There is increasing interest in the use of semiconductor diode lasers (780-800 nm) as Raman light sources.
Raman scattering vibrational spectral bands in solids are usually 5-20 cm.sup.-1 wide. An instrument must be able to resolve these bands and to observe very faint signals. The low efficiency of Raman scattering means that the collected signal may be only a few photons per second, although it is more commonly on the orders of 100-10,000 photons per second.
Like infrared spectra, Raman spectra are sensitive to small changes in molecular structure and are, therefore, reasonably good "fingerprints" for a molecule. A microscope which generates images of Raman scattering would be expected to be useful in many areas of science and technology. Materials which are commonly examined by Raman scattering include, but are not limited to: minerals, superconductors, semiconductors, polymers of all kinds, and biological materials including bones and teeth. An imaging system capable of imaging on two or more frequencies simultaneously would be especially useful for mapping heterogeneous materials.
The system according to the present invention provides the ability to image on many frequencies simultaneously. In principle, with the present invention it is possible to image on every band which is resolved by a spectrometer and viewed by the two-dimensional detector used. In practice, most applications would require imaging on 1-10 bands, because most microscopic samples contain relatively few components.
Photography has been used for many years to make permanent records of images generated under light microscopes. Since about 1980 video cameras have been attached to microscopes to generate images which can be recorded on video tape, or digitized and stored in a computer. If spectroscopic resolution is needed, the common practice is to put a filter or filters in the system to isolate the desired wavelengths. Such filters, which are customarily constructed of colored glass or multi-layer thin films (interference filters) isolate relatively broad bands, and are not suitable for Raman scattering or other high resolution techniques. Colored glass filters are further not tunable thus requiring a multitude of filters, one for each frequency band of interest. Interference filters are only tunable over a very limited range by rotating them perpendicular to the direction of propagation of light. Imaging at different wavelengths is accomplished by placement of different filters in the optical system. Filter systems are widely used in fluorescence microscopy and spectroscopically resolved visible light microscopy.
It is also possible to use interferometers as tunable filters in microscope imaging systems. Both Michelson interferometers and Fabry-Perot interferometers have been used for this purpose. Because infrared imaging detectors are unsatisfactory for infrared spectroscopy, the interferometer functions as a filter and point by point scanning is used to generate an image.
Although it is theoretically possible to use filters as a means of generating spectrally resolved images of Raman scattering, such systems would be generally unsatisfactory. Individual filters would be needed for each frequency and bandwidth desired. Also, it is often unknown which wavelengths are of interest in a sample without using a spectrograph to generate spectra for a sample. The present invention includes elements which enable the system to be used as a Raman point microprobe, enabling spectral characteristices of interest to be identified before attempting to generate a spectrally resolved image.
Raman spectra have been obtained under microscopes since about 1975. An early imaging device, called a MOLE (Molecular Optics Laser Examiner) was marketed for a few years. It was a low spectroscopic resolution system in which the entire magnified image was introduced into the spectrograph and viewed on a video monitor. The image was generated by a laser beam which was systematically scanned across the sample. The instrument was not very successful and has been withdrawn from the market. Point microprobes have been successful and have been continuously available since the middle 1970's. They are available from most or all of the major manufacturers of Raman spectrometers world wide.
The most common presently available Raman microscope imaging technique is to systematically move a sample on the stage of a microscope under a point-focused laser. This technique requires 30-90 minutes to generate an image, which is typically no finer than 100x100 pixels and has a spatial resolution determined by the laser focus, which is typically 1-3 microns. Similar techniques are used in infrared spectroscopy and require even longer times.
The system of the present invention utilizes means for illuminating a sample and collecting scattered light which is encoded and dispersed using a spectrometer. The dispersed image is projected onto a two-dimensional detector. Because it is intended for use with a video detector, one dimension of the detector is available for imaging the spatial dimension of the image which is parallel to the entrance slit of the spectrometer. Spatial image information perpendicular to the spectrometer entrance slit is obtained by using spatial encoding such as one-dimensional coded apertures (Hadamard masks). A sequence of encoded images are serially line-focused into the spectrograph which provides for efficient spectral resolution without loss of information in the spatial dimension parallel to the slit. From the sequences of spectroscopically resolved line images, two-dimensional spectroscopically resolved images can be recovered using a reverse transform function.
Hadamard multiplexing was developed for spectroscopy and imaging in the period of 1968 to 1975. In the spectroscopic application a series of Hadamard masks encodes the dispersed radiation at the exit plane of a spectrograph for recording on a single channel detector, which sums the signals from the various open apertures. The spectrum can be recovered from the sequence of encoded signals. In addition, the entrance slit of the spectrograph can be replaced by a series of coded apertures, thus increasing the through-put of the instrument. Hadamard imaging was developed during the same interval. In this application, the image is brought to a focus on a two-dimensional Hadamard mask system, which encoded spatial information for detection by a single channel detector. Occasionally, one-dimensional images only were obtained by this technique using one-dimensional encoding. The spatially resolved images could also be brought to a Hadamard spectrometer, although this technique did not prove practical.
It is also possible to encode the light source to generate spatially resolved images. This technique was proposed in the 1970's and has been used to obtain photoacoustic and photothermal images.
With suitable illumination, the system of the present invention can operate in any of several microscopy modes, including transmission, reflection, fluorescence and light scattering. The system is capable of acquiring images at multiple wavelengths simultaneously and is especially suitable for obtaining faint images with high spectroscopic resolution. For this reason the system of this invention is ideally suited in imaging materials for their Raman light scattering properties.
The system of this invention achieves an average analysis time advantage because it allows illumination with much higher laser intensity than is possible with conventional, point-focused, Raman microprobes. Consequently, stronger signals are generated, and these require shorter acquisition times. High illumination power can be used because the source illuminates the entire sample, thus decreasing the local light intensity incident on the sample, thereby minimizing the probability of sample thermal damage.
Additional benefits and advantages of the present invention will become apparent to those skilled in the art to which this invention relates from the subsequent description of the preferred embodiments and the appended claims, taken in conjunction with the accompanying drawings.